The Problem: Medical management of many critically ill patients necessitates delivery of high concentrations of inspired oxygen to maintain tissue oxygenation. However, hyperoxic therapy creates a dilemma as it directly damages cells. While extensive research has implicated superoxide anion (O2-) or O2- derived products as key mediators of hyperoxic injury, the precise locations of O2- generation and its specific molecular targets are not known. Our hypothesis is that mitochondrial electron transport contributes to increased O2- production in hyperoxia and that O2- damages both mitochondrial and cytosolic targets. We have chosen a yeast model to initially test our hypothesis. Yeast are eukaryotic cells with important similarities to mammalian cells. However, unlike mammalian cells, yeast can be mutated in precise ways and can grow facultatively if electron transport is disrupted. Our preliminary data support our premise. First, we found that yeast which lack the mitochondrial form of superoxide dismutase (Mn-SOD) are sensitive to hyperoxia despite normal levels of the cytosolic form of SOD (Cu,Zn- SOD). Oxygen sensitivity is increased if electron transport activity is increased with non-fermentable carbon sources. Complete genetic disruption of electron transport in the Mn-SOD deficient cell restores oxygen resistance. These data strongly implicate mitochondrial electron transport as an important source of O2- in hyperoxia. Second, we found that in enriched media with glucose, yeast lacking Cu,Zn-SOD but not Mn-SOD grow normally in room air but fail to grow when electron transport is increased by supplying a non-fermentable carbon source in place of glucose. These data imply that O2- generated in the mitochondria leaks into and causes damage in the cytosol. My immediate specific objectives are to identify a) the relevant source(s) of O2- in the electron transport chain, b) the magnitude of O2- release from mitochondria in hyperoxia, c) the molecular targets of O2- within mitochondria, d) the role of Cu,Zn-SOD in scavenging O2- released from mitochondria, e) if mitochondria can be uncoupled by back diffusion of protonated O2- and f) if extramitochondrial SOD can protect the mitochondria from hyperoxic damage. The significance of this project is to provide new information about the role of mitochondrial electron transport in hyperoxic injury. The site(s) of O2- production in the electron transport chain and its principal molecular targets must be identified in order to better understand how O2- mediates hyperoxic injury and how the poor outcome of patients with catastrophic illnesses who require hyperoxic therapy can be improved.